Ion channels underlie all electrical excitability in the brain and heart, and defects in ion channels are the cause of many human disorders. The KCNH family of channels, ERG, ELK, and EAG, are voltage-dependent potassium channels that are specialized for their role in shaping the electrical activity of neurons and cardiomyocytes. Defects in KCNH channels have been shown to be linked to cognitive defects, increased risk of schizophrenia, cardiac arrhythmias, and cancer. Recently it has become clear that the specialized gating of KCNH channels arises from their unique intracellular domains, the N-terminal eag domain, and the C-terminal C-linker/CNBHD. In this proposal, we will test the hypothesis that the C-linker/CNBHD of KCNH channels is a key regulatory domain that mediates the action of the eag domain and other intracellular regulators. We will build on three exciting new X-ray crystal structures we have solved of the intracellular domains of three different KCNH channels: 1) the C-linker/CNBHD of ELK, 2) the C-linker/CNBHD of ERG, and 3) a complex of the CNBHD and eag domain of EAG. These new structures reveal unexpected features like a direct interaction between the eag domain and the CNBHD, and an intrinsic ligand in the binding site of the CNBHD that regulates gating. They provide a framework to better understand the mechanisms of the gating and regulation of KCNH channels. Toward this goal, we will leverage the power of X-ray crystallography, electrophysiology, and fluorescence to study the structure and rearrangements of the eag domain and C-linker/CNBHD during gating of KCNH channels.